Determination of galantamine hydrobromide in bulk drug and pharmaceutical dosage form by spectrofluorimetry.

AIM: To develop a simple, accurate, sensitive, rapid and precise method for the determination of galantamine hydrobromide in bulk drug and pharmaceutical dosage form.
MATERIAL AND METHODS: The method employs wavelength detection and determination of galantamine hydrobromide at excitation wavelength 282 nm and emission wavelength 607 nm in a solution of simple distilled water. RESULT AND
CONCLUSION: The method was found to be linear in the range of 2-14 μg/ml having r (2) = 0.9999. The mean accuracy was found to be 98.12% to 99.67%. The intraday and interday precision was found to be 0.18-0.35% and 0.13-0.46%, respectively. The limit of detection was found to be 0.29 μg/ml. The limit of quantification was found to be 0.89 μg/ml. The method was successfully applied for the determination of galantamine hydrobromide in bulk drug as well as pharmaceutical dosage form.

Galantamine hydrobromide is a reversible, competitive acetyl cholinesterase inhibitor. It is known chemically as (4aS, 6R, 8aS)-4a, 5, 9, 10, 11, 12-hexahydro-3-methoxy-11-methyl-6H-benzofuro [3a, 3,2-ef][2] benzazepin-6-ol hydrobromide. It has an empirical formula of C17H21NO3 HBr and a molecular weight of 368.27. Galantamine hydrobromide is indicated for the treatment of mild to moderate dementia of the Alzheimer's type. Galantamine hydrobromide is a white to almost white powder and is sparingly soluble in water. The structural formula for galantamine hydrobromide is shown in Figure 1. There is no any official method for determination of galantamine hydrobromide. Different analytical methods like HPLC,[1] micellar electrokinetic chromatography–electrospray ionization mass spectrometry,[2] high-performance liquid chromatographic method with UV photodiode array, fluorescence and mass spectrometric detection,[3] RP-HPLC[4] liquid chromatographic-tandem mass spectrometric method[56] and Spectrofluorimetry,[7] were reported for determination of galantamine hydrobromide in biological fluids as well as in plants. Although these techniques are sufficiently sensitive, most of them use expensive instruments or are somewhat tedious and time-consuming. The present spectrofluorimetric method is simple, accurate and sensitive. Also, the present spectrofluorimetric method uses a simple solvent and does not require complicated sample preparation. Therefore, the aim of this study was to develop and validate fast, economic, and selective methods for a routine quality control analysis of pharmaceutical product.

Figure 1

Chemical structure of galantamine hydrobromide

Materials and Methods

Chemicals and reagents

Pure samples: Galantamine hydrobromide was kindly supplied by Cadila Pharmaceutical Ltd. (Ahmedabad, India)

Market samples: Galamer 4 mg tablets (Sun Pharmaceutical ltd.)

Chemical and Reagents: Distilled water prepared by distillation unit in the laboratory was used throughout the study.[8910111213]

Apparatus and instruments

Spectrofluorimeter, Model RF 1501(Shimadzu)

Balance, Model ALC 210.4 (Acculab)

Ultra Sonicator (Fast Clean Ultrasonic Cleaner)

Volumetric flasks – 10 ml, 100 ml

Pipettes – 1 ml, 5 ml, 10 ml, beakers, measuring cylinders etc.

Standard solutions

Stock solutions: Galantamine hydrobromide stock solution was prepared by weighing accurately 100 mg of galantamine hydrobromide powder transferred into a 100 ml volumetric flask; 50 ml distilled water was added, shaken for a few minutes, and diluted to volume with distilled water to get 1000 μg/ml concentration, sonicated for 3 min. This solution was used as a working standard solution.

Working solutions: For spectrofluorimetry, 10 ml of the stock solution was transferred into the 100 ml volumetric flask and diluted to the mark with distilled water to get final concentration of 100 μg/ml.

Wavelength set

For the wavelength detection the working standard solution of Galantamine hydrobromide was scanned in spectroflourimeter and got 282 nm as excitation wavelength and 607 nm as the emission wavelength.

Procedures for validation parameters

Linearity

From the working solution (100 μg/ml), aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 ml were transferred to a series of 10 ml volumetric flasks. The volume was adjusted to a mark with distilled water to get 2, 4, 6, 8, 10, 12, and 14 μg/ml of solution. The fluorescence intensity was measured at 282 nm as excitation wavelength and 607 nm as the emission wavelength over the concentration range of 2–14 μg/ml and was plotted against concentration and regression equation was calculated.

Precision

Repeatability (Precision on replication):

From the working solution (100 μg/ml), aliquot of 0.6 ml was transferred to the 10 ml volumetric flask and diluted up to mark with distilled water (6 μg/ml). The fluorescence intensity of this solution was measured at 282 nm as excitation wavelength and 607 nm as emission wavelength. The fluorescence intensity of the same solution was measured five times and % CV was calculated.

Intraday and Interday Precision:

Intraday precision was determined by analyzing galantamine hydrobromide (4, 8 and 12 μg/ml) for three times in the same day and % CV was calculated.

Interday precision was determined by analyzing galantamine hydrobromide (4, 8 and 12 μg/ml) daily for 5 days and % CV was calculated.

Accuracy

It was determined by calculating the recovery of galantamine hydrobromide by standard addition method. To a fixed amount of galantamine hydrobromide (6 μg/ml) of pre-analyzed sample of galantamine hydrobromide, increasing amount of galantamine hydrobromide was added at all levels of calibration curve and the amount of galantamine hydrobromide was calculated at each level.

Limit of Detection

Based on visual evaluation: It is determined by the analysis of samples with known concentrations of analyte and establishing the minimum level at which the analyte can be reliably detected.

Based on signal to noise ratio: Determination of the signal to noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal to noise ratio of 2:1 or 3:1 is generally considered acceptable for estimating the detection limit.

Based on standard deviation of the response and the slope: The detection limits may be expressed as:

LOD = 3.3 s / S

where, s = the standard deviation of the response

S = the slope of calibration curve

Limit of Quantification

Based on visual evaluation: It is determined by the analysis of samples with known concentrations of analyte and establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision

Based on signal to noise: Determination of the signal to noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably quantified. A signal to noise ratio of 10:1 is generally considered acceptable for estimating the quantitation limit.

Based on standard deviation of the response and the slope: The quantitation limits may be expressed as:

LOQ = 10 s / S where,

s = the standard deviation of the response

S = the slope of calibration curve

Application to pharmaceutical preparation

To determine the content of galantamine hydrobromide in tablet (label claim: 4 mg/tablet), the contents of 20 tablets were weighed and their mean weight determined and finely powdered. An equivalent weight of the tablet content was transferred into a 100 ml volumetric flask containing 50 ml distilled water, sonicated for 30 min and diluted to 100 ml with distilled water. The resulting solution was sonicated for 30 min and supernatant was filtered through Whatman filter paper. 0.6 ml from this solution was transferred to 50 ml volumetric flask and diluted with distilled water to get 6 μg/ml concentrations. The fluorescence intensity was measured at 282 nm as excitation wavelength and 607 nm as the emission wavelength and concentration of sample solution was found from calibration curve of galantamine hydrobromide.

Results and Discussion

This method, involved determination of galantamine hydrobromide, by measuring the fluorescence intensity at excitation wavelength 282 nm and emission wavelength 607 nm. The method was found to be linear in the range of 2–14 μg/ml having r2 = 0.9999 [Figure 2, Table 1]. The mean accuracy was found to be 98.12% to 99.67% [Table 2]. The intraday and interday precision was found to be 0.18–0.35% and 0.13–0.46%, respectively [Table 3]. The limit of detection was found to be 0.29 μg/ml. The limit of quantification was found to be 0.89 μg/ml. The validity of the method was further checked by applying the standard addition method. Results indicate that the proposed method is valid and applicable for the determination of galantamine hydrobromide in pharmaceutical formulation [Tables 4 and 5].

Figure 2

Calibration curve of galantamine hydrobromide by spectrofluorimetry. Y = m X + c, where X is the concentration of drug in μg/ml, Y is the fluorescence intensity measured at 282 nm as excitation wavelength and 607 nm as the emission wavelength, m is slope, and c is intercept

Table 1

Calibration data of standard galantamine hydrobromide by spectrofluorimetry

Table 2

Accuracy data of galantamine hydrobromide by spectrofluorimetry

Table 3

Intraday and interday precision data of galantamine hydrobromide by spectrofluorimetry

Table 4

Estimation of galantamine hydrobromide in tablet by spectrofluorimetry

Table 5

Summary of validation parameters by spectrofluorimetry

Conclusion

According to ICH Guideline Q2 (R1), the proposed method was simple, accurate, precise, sensitive and reproducible. The statistical results of the analysis of the tablets by this method were reproducible, reliable, and were in good agreement with labeled claim of the drug. There was no interference of the present excipients in the tablets. The proposed procedures can be applied for the determination of galantamine hydrobromide in pharmaceutical formulation.